The general goal of this project is to understand patterning and morphogenesis during vertebrate embryogenesis, using Sonic hedgehog (Shh) signaling in the limb bud as a model. Building on the results of the previous five years of funding, we have three Specific Aims. The First makes use of an extremely powerful in vitro system we have developed where we can maintain limb bud cells in an undifferentiated state for several weeks and still reconsitute them as a limb bud and assess their morphological potential. We will use this to study the integration of time and dose in establishing threshold responses to Shh activity in patterning the limb and also study how various signals work together to define proximodistal limb pattern. A second Aim focuses on the role of Shh in controlling the size of the limb bud and its synergistic activity with the Hippo/YAP pathway. This part of the project will be carried out in mice, using activatable and conditionally deficient alleles of YAP. Mice harboring these alleles will be bred to lines of mice expressing cre recombinase exclusively in the Shh-expressing cells, throughout the limb bud, and in the limb myoblasts, thereby activating or inactivating the YAP/HIPPO pathway specifically in these tissues. Finally, a third Aim will address the important, but poorly understood phenomonom of scaling, wherein the same signaling systems pattern very small or large embryos in an equivelant manner. We will focus on themechanisms interpreting Shh signaling in the relatively large limb bud of the chick in comparison to the relatively small limb bud of the direct developing frog coqui.